Technical Tips

Total Phosphorus Procedure

Determination of Phosphates in Wastewater
(adapted from procedure 4500-P B and 4500-P E, Standard Methods, 21st edition)

Notes before proceeding:

1. This procedure is based on the above reference procedures. There is no change in the chemistry; the only changes are in volumes. The Standard Methods procedure has you start out with 50 ml, digest, bring the volume up to 100 ml, take 50 ml of that, adjust the pH, and add 8 ml of combined reagent, giving you a final volume of about 58 ml. This means that:
   - The 0.1 ppm standard actually ends up being 0.0431 ppm
   - The 0.2 ppm standard actually ends up being 0.0862 ppm
   - The 0.5 ppm standard actually ends up being 0.2155 ppm, etc.
   Throw in a dilution factor, and this is one reason why so many people have trouble with this test.
   This procedure has you start with 50 ml, digest, adjust the pH, add 8 ml of combined reagent, and bring the final volume back up to 50 ml. This means that:
   - The 0.1 ppm standard actually ends up being 0.1 ppm
   - The 0.2 ppm standard actually ends up being 0.2 ppm
   - The 0.5 ppm standard actually ends up being 0.5 ppm, etc.
2. The hot plate digestion reduces sample volumes to approximately 20 ml, leaving plenty of room to adjust the pH, and add the combined reagent, before bringing the final volume back up to 50 ml. This procedure may not work with an autoclave digestion, since it does not reduce the volume appreciably. If using an autoclave, you may have to adjust the pH, bring the volume up to 50 ml, then add 8 ml of combined reagent for a total volume of 58 ml. See note after step #7 in the following procedure.
3. The Wisconsin DNR has stated that they want a "linear regression" or "least square" calculation for the "curve", instead of a graphical solution. This can easily be done with many calculators or with a computer program. However, we highly recommend that you initially run standards up to 1.0 ppm as P if you are using this procedure, or 2.0 ppm if you are using the Standard Methods' procedures. Then plot the results graphically to determine the "A" maximum value for which the graph is linear. You CANNOT use "A" readings for samples above this value and expect to get the right answer!

I. Preparation of Reagents
Note: We supply most of the reagents already made up. However, the Ascorbic Acid solution is only stable for 1 week, and the combined reagent is only stable for 4 hours. We recommend that these two reagents be prepared fresh daily.

1. 5 N Sulfuric Acid. We sell this made up.
2. Potassium Antimonyl Tartrate Solution. Fill a 500 ml volumetric flask about half full with distilled water. Weigh out 1.3715 grams of Potassium Antimonyl Tartrate on an analytical balance and transfer to the flask. Mix to dissolve. Fill flask to the 500 ml mark with distilled water and mix thoroughly. Store in a glass stoppered bottle.
3. Ammonium Molybdate Solution. Pour about 400 ml of distilled water into a 600 ml or 1000 ml beaker. Add a stir bar and place on a stir plate. Add 20 grams of Ammonium Molybdate and stir until dissolved. Transfer to a 500 ml volumetric flask, fill to the mark, and mix thoroughly. Store in a glass stoppered bottle.
4. Ascorbic Acid Solution. Prepare fresh daily. Place about 70 ml of distilled water in a 150 ml beaker. Add a stir bar and place on a stir plate. Weigh out 1.7600 grams of Ascorbic Acid on an analytical balance. Transfer to beaker and stir until dissolved. Transfer to a 100 ml volumetric flask, fill to the mark, and mix thoroughly. Discard when it turns brown.
5. Combined reagent. Must be prepared fresh, just before using. Let all reagents come to room temperature before mixing. Mix, in order, with constant stirring:
   - 50 ml of 5 N Sulfuric Acid
   - 5 ml of Potassium Antimonyl Tartrate Solution
   - 15 ml of Ammonium Molybdate Solution
   - 30 ml of Ascorbic Acid Solution
6. Phosphate Standard, 5 ppm as P. Pipet 50 ml of 50 ppm Phosphate Standard into a 500 ml volumetric flask, dilute to the mark, and mix thoroughly.

II. Preparation of Standard Curve

1. Using 125 ml Erlenmeyer flasks, prepare blank, standards, and samples as follow:
   

   Flask #	ml of 5 ppm standard	final conc/as P
   1	0	0.000 (blank)
   2	1	0.100 ppm
   3	2	0.200 ppm
   4	3	0.300 ppm
   5	4	0.400 ppm
   6	5	0.500 ppm
   7	8	0.800 ppm
   8, etc.	0	????

   
   Note: Flasks 8, etc., will have 50.0 ml (or some other volume-measured exactly) of sample to be tested, and no standard. If the readings come out greater than 0.8 ppm, the sample will have to be diluted with distilled water and re-run. THEREFORE YOU SHOULD RUN SEVERAL DIFFERENT DILUTIONS FOR SAMPLES, AND ESPECIALLY FOR REFERENCE STANDARDS.
2. Fill each erlenmeyer flask approximately up to the 50 ml mark. This does not have to be exact. The samples need to be measure exactly, using volumetric pipets.
3. Add 2-3 drops of Phenolphthalein to each. If a pink color develops, add enough 11 N Sulfuric Acid to just discharge the color. Add 1 ml excess 11 N Sulfuric Acid to each erlenmeyer flask.
4. Add 0.4 g of Ammonium Persulfate OR 0.5 g of Potassium Persulfate to each. Add an inert boiling bead or boiling stone to each flask.
5. Place all flasks on a hot plate and gently boil down to a volume of 10 to 20 ml.
6. Add 2-3 drops of Phenolphthalein to each, and add Sodium Hydroxide Solution (6 N) to each, drop wise, until a faint pink color develops. Then add 5 N Sulfuric Acid drop wise to turn the solution clear again.
7. If using a hot plate for digestion, transfer the contents of each erlenmeyer to a 50 ml volumetric flask. Triple-rinse each erlenmeyer with a small quantity of distilled water to ensure a quantitative transfer. Pipet 8 ml of freshly prepared Combined Reagent to each and bring to the mark with distilled water. Mix well.
   * Note: If using an autoclave for digestion, there may not be room to add the Combined Reagent and still keep the total volume below 50 ml. If this is the case, bring the total volume up to 50 ml in the volumetric flasks, then add 8 ml of the Combined Reagent to each flask. If you do this with every standard and every sample, the dilution factor caused by adding the Combined Reagent will cancel out, and you can still plot "Absorbance" against "mg/l as P".
8. Take color measurements at least 10 minutes but no more than 30 minutes after Step 7 is completed.
9. Set the instrument to the "T" mode (Transmittance). Follow manufacturer's instructions to Zero the instrument.
10. Transfer the contents of the blank to a cuvet. Place the blank into the cuvet holder of the instrument and adjust for 100% T.
11. With the blank still in place, switch the instrument to the "A" mode (Absorbance). The reading should go to 0.000 or .000. If it does not, adjust for 0.000.
12. Transfer the contents of each flask to a cuvet. Place each cuvet into the holder.
13. Record the reading in the "A" mode (Absorbance) for each standard.
14. Prepare a graph on linear graph paper with the "Absorbance" reading on the vertical axis and the "mg/l as P" on the horizontal axis to determine whether you have exceeded the linear portion of the curve.
15. Using a calculator or computer program, calculate the slope and intercept for the linear portion of the graph by doing a linear regression. The equation will take the form of: A = mP + b
    The calculation will determine the values for "m" and "b". Once these are determined, just plug in the "A" value of your samples, and this will give you "P", which is "mg/l as P". Remember to multiply by the dilution factor for the sample, if applicable.

III. Daily Routine Analysis for Phosphates

1. Prepare one blank, one standard (in the range that you expect the sample to be) and your unknown samples as in Step 1, Section II.
2. Follow Steps 2 through 12 in Section II.
3. Use the "A" value for your standard in the equation to calculate the "mg/l as P" in the standard. If this value is within 10% of the true value, proceed to Step 4 below. If it is not within 10% of the true value, the standard curve needs to be re-run.
4. Place the unknown samples in the unit and record the "A" readings. Use these values in your equation to calculate the "mg/l as P" for each sample. Remember to multiply by the dilution factors, if applicable, to get your final answer.

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